This Program Project application requests continued funding of research the unifying theme of which is cellular and molecular biology of normal and abnormal erythroid membranes. The Program Project has six component projects and three supporting cores. It has four major research objectives: (a) Characterization of the regulatory elements of the band 3 gene and studies of the expression of membrane protein genes during erythroid differentiation. This work is the main focus of Project 1 (K. Sahr, Erythroid band 3 gene regulatory elements) and Project 2 (M. Hanspal, Biogenesis of normal and abnormal erythrocyte membrane); (b) Characterization of molecular defects of membrane proteins in hereditary elliptocytosis and spherocytosis with principal effort directed to identification at the DNA level, of alpha and beta spectrin mutations causing defective self-association of spectrin dimer to tetramers in hereditary elliptocytosis, as well as molecular defects of ankyrin and band 3 protein in hereditary spherocytosis and Southeast Asian ovalocytosis, principal effort of Project 3 (J. Palek, Molecular defects of membrane proteins) and, in part, Project 2 (M. Hanspal, Biogenesis of normal and abnormal erythrocyte membrane), and Project 5 (S.C. Liu, Band 3 protein in Southeast Asian ovalocytosis); (c) Red cell membrane protein reorganization during invasion and release of malaria parasites with emphasis on the role of the band 3 protein in the entry of the parasites into the cells and the contribution of phosphorylation of the 4.1 protein and proteolytic degradation of the 4.1 protein and ankyrin in facilitating the release of the parasites from erythrocytes (main focus of Project 4 (A. Husain- Chishti, Membrane skeleton of P. falciparum infected erythrocytes) and Project 5 (S.C. Liu, Band 3 protein in Southeast Asian ovalocytosis); and (d) Structural and functional characterization of red cell membrane proteins, with focus on the cellular and molecular biology of 4.2 protein (Project 6, C. Cohen, Structural and functional studies of band 4.2). While in the previous funding period, our major focus was directed to membrane skeleton and its disorders, band 3 protein emerges as an additional area of joint focus in the competing renewal application: both in terms of regulation of band 3 protein expression and role in membrane biogenesis (Projects 1 and 2), function and molecular defects (Projects 3, 5, and 6) and regulation of cell biomechanical properties and malaria parasite invasion (Projects 3, 4, and 5). Although many areas of expertise in the field of cell and molecular biology of red cell membrane are common to all members of the Program Project, there are specific areas of expertise which are unique to the individual projects. They will be utilized to strengthen, through a close collaboration, the overall quality of the Program Project. They include (a) Molecular biology of normal and abnormal membrane protein genes, (b) Erythroid cell culture, (c) Protein purification and structural characterization, (d) Electron microscopy, (e) Malaria culture techniques, and (f) Clinical and laboratory diagnostic expertise.